The invention refers to yeast strains transformed with at least one copy of a gene coding for lactic dehydrogenase (LDH) and further modified for the production of lactic acid with high yield and productivity.
The applications of lactic acid and its derivatives encompass many fields of industrial activities (i.e., chemistry, cosmetic, and pharmacy), as well as important aspects of food manufacture and use. Furthermore, today there is growing interest in the production of such an organic acid to be used directly for the synthesis of biodegradable polymer materials.
Lactic acid may be produced by chemical synthesis or by fermentation of carbohydrates using microorganisms. The latter method is now commercially preferred because microorganisms have been developed that produce exclusively one isomer, as opposed to the racemic mixture generated by chemical synthesis. The most important industrial microorganisms, such as species of the genera Lactobacillus, Bacillus, and Rhizopus, produce L(+)-lactic acid. Production by fermentation of D(xe2x88x92)-lactic acid or mixtures of L(+)- and D(xe2x88x92)-lactic acid are also known.
During a typical lactic acid fermentation, there is an inhibitory effect caused by lactic acid produced on the metabolic activities of the producing microorganism. Besides the presence of lactic acid, lowering the pH value also inhibits cell growth and metabolic activity. As a result, the extent of lactic acid production is greatly reduced.
Therefore, the addition of Ca(OH)2, CaCO3, NaOH, or NH4OH to neutralise the lactic acid and to thereby prevent the pH decrease is a conventional operation in industrial processes to counteract the negative effects of free lactic acid accumulation.
These processes allow the production of lactate(s) by maintaining the pH at a constant value in the range of about 5 to 7; this is well above the pKa of lactic acid, 3.86.
Major disadvantages are connected to the neutralisation of lactic acid during the fermentation. Mainly, additional operations are required to regenerate free lactic acid from its salt and to dispose of or recycle the neutralising cation; this is an expensive process. All the extra operations and expense could be eliminated if free lactic acid could be accumulated by microorganisms growing at low pH values, thus minimising the production of lactate(s).
It has been proposed the use of recombinant yeasts expressing the lactate dehydrogenase gene so as to shift the glycolytic flux towards the production of lactic acid.
FR-A-2 692 591 (Institut Nationale la Recherche Agronomique) discloses yeast strains, particularly Saccharomyces strains, containing at least one copy of a gene coding for a lactate dehydrogenase from a lactic bacterium, said gene being under the control of sequences regulating its expression in yeasts.
Said strains may give both the alcoholic and the lactic fermentation and this so called xe2x80x9cintermediatexe2x80x9d or xe2x80x9cbalancedxe2x80x9d fermentation could be exploited in areas such as brewing, enology, and baking.
Porro et al., (Biotechnol. Prog. 11, 294-298, 1995) have also reported the transformation of S. cerevisiae with a gene coding for bovine lactate dehydrogenase.
However, because of the high production of ethanol, the yield in the production of lactic acid for both the processes described was not considered to be competitive with that obtainable by the use of lactic bacteria.
In the past decade, xe2x80x9cnon conventional yeastsxe2x80x9d other than S. cerevisiae have gained considerable industrial interest as host for the expression of heterologous proteins. Examples are the methanol-utilising yeasts such as Hansenula polimorpha and Pichia Pastoris, the lactose-utilizing yeasts such as Kluyveromyces lactis. In addition to enabling the use of a wider range of substrates as carbon and energy sources, other arguments have been put forward to the industrial use of xe2x80x9cnon conventional yeastsxe2x80x9d. Generally speaking, biomass and product-yield are less affected, in some of these yeasts, by extreme conditions of the cellular environment. High-sugar-tolerant (i.e., 50-80% w/v glucose medium; Torulaspora-syn. Zygosaccharomyces-delbrueckii, Zygosaccharomyces rouxii and Zygosaccharomyces bailii; Ok T and Hashinaga F., Journal of General and Applied Microbiology 43(1): 39-47, 1997) and acid- and lactic-tolerant (Zygosaccharomyces rouxii and Zygosaccharomyces bailii; Houtsma P C, et al., Journal of Food Protection 59(12), 1300-1304, 1996.) xe2x80x9cnon conventional yeastsxe2x80x9d are available. As already underlined the cost of down stream processing could be strongly reduced if the fermentation process is carried out under one or more of the above mentioned xe2x80x9cextreme conditionsxe2x80x9d.
According to a first embodiment, this invention provides yeast strains lacking ethanol production ability or having a reduced ethanol production ability and transformed with at least one copy of a gene coding for lactic dehydrogenase (LDH) functionally linked to promoter sequences allowing the expression of said gene in yeasts.
More particularly, this invention provides yeast strains having a reduced pyruvate dehydrogenase activity and a reduced pyruvate decarboxylase activity and transformed with at least one copy of a gene coding for lactic dehydrogenase (LDH) functionally linked to promoter sequences allowing the expression of said gene in yeasts.
According to another embodiment, this invention provides yeast strains of Kluyveromyces, Torulaspora and Zygosaccharomyces species, transformed with at least one copy of a gene coding for lactic dehydrogenase (LDH) functionally linked to promoter sequences allowing the expression of the gene in said yeasts.
According to a further embodiment, the invention also provides yeast cells transformed with a heterologous LDH gene and overexpressing a lactate transporter.
Other embodiments are the expression vectors comprising a DNA sequence coding for a lactic dehydrogenase functionally linked to a yeast promoter sequence and a process for the preparation of DL-, D- or L-lactic acid by culturing the above described metabolically engineered yeast strains in a fermentation medium containing a carbon source and recovering lactic acid from the fermentation medium.
Furthermore, the invention provides processes for improving the productivity (g/l/hr), production (g/l) and yield (g/g) on the carbon source of lactic acid by culturing said yeast strains in a manipulated fermentation medium and recovering lactic acid from the fermentation medium.
It has been found that production of lactic acid can be obtained by metabolically modified yeasts belonging to the genera Kluyveromyces, Saccharomyces, Torulaspora and Zygosaccharomyces.
More particularly, it has been found that very high yields in the production of lactic acid are obtained by engineered yeast strains so as to replace at least the ethanolic fermentation by lactic fermentation.
Even higher yields ( greater than 80% g/g) in the production of lactic acid may be obtained by engineered yeast strains so as to replace both the ethanolic fermentation and the use of pyruvate by the mitochondria by lactic fermentation.
To this purpose, the invention also provides transformed yeast cells having an increased LDH activity, for instance as a consequence of an increased LDH copy number per cell or of the use of stronger promoters controlling LDH expression.
An increased LDH copy number per cell means at least one copy of a nucleic acid sequence encoding for lactic dehydrogenase protein, preferably at least two copies, more preferably four copies or, even more preferably, at least 10-50 copies of said nucleic acid sequence.
In order to have the highest production of lactic acid, yeast cells transformed according to the invention preferably overexpress a lactate transporter. This can be obtained by transforming yeast cells with one or more copies of a gene required for lactate transport.
The strains according to the invention can be obtained by several methods, for instance by genetic engineering techniques aiming at the expression of a lactate dehydrogenase activity, and by inactivating or suppressing enzymatic activities involved in the production of ethanol, e.g. pyruvate decarboxylase and alcohol dehydrogenase activities, and by inactivating or suppressing enzymatic activities involved in the utilisation of pyruvate by the mitochondria.
Since pyruvate decarboxylase catalyses the first step in the alcohol pathway, yeast strains without or having a substantially reduced pyruvate decarboxylase (PDC) activity and expressing a heterologous lactate dehydrogenase gene are preferred.
Further, since pyruvate dehydrogenase catalyzes the first step in the utilization of pyruvate by the mitochondria, yeast strains having no or a substantially reduced pyruvate dehydrogenase (PDH) activity and expressing a heterologous lactate dehydrogenase gene are also preferred.
Since lactate is excreted in the medium via a lactate transporter, cells producing lactic acid and verexpressing the lactate transporter are also preferred.
The expression of a LDH gene in yeast strains allows the production of lactic acid at acid pH values so that the free acid is directly obtained and the cumbersome conversion and recovery of lactate salts are minimized. In this invention, the pH of the fermentation medium may initially be higher than 4.5, but will decrease to a pH of 4.5 or less, preferably to a pH of 3 or less at the termination of the fermentation.
Any kind of yeast strain may be used according to the invention, but Kluyveromyces, Saccharomyces, Torulaspora and Zygosaccharomyces species are preferred because these strains can grow and/or metabolize at very low pH, especially in the range of pH 4.5 or less; genetic engineering methods for these strains are well-developed; and these strains are widely accepted for use in food-related applications.
Good yields of lactic acid can moreover be obtained by Kluyveromyces, Torulaspora and Zygosaccharomyces strains transformed with a gene coding for lactic dehydrogenase having a xe2x80x9cwild-typexe2x80x9d pyruvate decarboxylase and/or a pyruvate dehydrogenase activity.
The term xe2x80x9creduced pyruvate decarboxylase activityxe2x80x9d means either a decreased concentration of enzyme in the cell or reduced or no specific catalytic activity of the enzyme.
The term xe2x80x9creduced pyruvate dehydrogenase activityxe2x80x9d means either a decreased concentration of enzyme in the cell or reduced or no specific catalytic activity of the enzyme.
According to the invention, it is preferred the use of strains wherein the ethanol production is or approaches zero but a reduced production for instance at least 60% lower, preferably at least 80% lower and even more preferably at least 90% lower than the normal of wild-type strains is acceptable.
According to the invention, it is preferred the use of strains wherein the pyruvate decarboxylase and/or pyruvate dehydrogenase activities are or approach zero but a reduced activity for instance at least 60% lower, preferably at least 80% lower and even more preferably at least 90% lower than the normal of wild-type strains is acceptable.
An example of K. lactis having no PDC activity has been disclosed in Mol. Microbiol. 19 (1), 27-36, 1996.
Examples of Saccharomyces strains having a reduced PDC activity are available from ATCC under Acc. No. 200027 and 200028. A further example of a Saccharomyces strain having a reduced PDC activity as a consequence of the deletion of the regulatory PDC2 gene has been described in Hohmann S (1993) (Mol Gen Genet 241:657-666).
An example of a Saccharomyces strain having no PDC activity has been described in Flikweert M. T. et al. (Yeast, 12:247-257, 1996). In S. cerevisiae reduction of the PDC activity can be obtained either by deletion of the structural genes (PDC1, PDC5, PDC6) or deletion of the regulatory gene (PDC2).
An example of Kluyveromyces strain having no PDH activity has been described in Zeeman et al. (Genes involved in pyruvate metabolism in K. lactis; Yeast, vol 13 Special Issue April 1997, Eighteenth International Conference on Yeast Genetics and Molecular Biology, p143).
An example of Saccharomyces strain having no PDH activity has been described in Pronk J T. et al. (Microbiology. 140 (Pt 3):601-10, 1994).
PDC genes are highly conserved among the different yeast genera (Bianchi et al., Molecular Microbiology, 19(1):27-36, 1996; Lu P. et al., Applied and Environmental Microbiology, 64(1):94-7, 1998). Therefore it can be easily anticipated that following classical molecular approaches, as reported by Lu P. et al. (supra), it is possible to identify, to clone and to disrupt the gene(s) required for a pyruvate decarboxylase activity from both Torulaspora and Zygosaccharomyces yeast species. Further, it can be also anticipated that following the same classical approaches, as reported by Neveling U. et al. (1998, Journal of Bacteriology, 180(6):1540-8, 1998), it is possible to isolate, to clone and to disrupt the gene(s) required for the PDH activity in both Torulaspora and Zygosaccharomyces yeast species.
The pyruvate decarboxylase activity can be measured by known methods, e.g. Ulbrich J., Methods in Enzymology, Vol. 18, p. 109-115, 1970, Academic Press, New York.
The pyruvate dehydrogenase activity can be measured by known methods, e. g. according to Neveling U. et al. (supra).
Suitable strains can be obtained by selecting mutations and/or engineering of wild-type or collection strains. Hundreds of mutants could be selected by xe2x80x9chigh throughput screenxe2x80x9d approaches. The modulation of pyruvate decarboxylase activity by using nutrients supporting different glycolytic flow rates (Biotechnol. Prog. 11, 294-298, 1995) did not prove to be satisfactory.
A preferred method for decreasing or destroying the pyruvate decarboxylase activity and/or pyruvate dehydrogenase activity in a yeast strain according to the invention consists in the deletion of the corresponding gene or genes.
These deletions can be carried out by known methods, such as that disclosed in Bianchi et al., (Molecular Microbiol. 19 (1),27-36, 1996; Flikweert M. T. et al., Yeast, 12:247-257, 1996 and Pronk J T. et al., Microbiology. 140 (Pt 3):601-10, 1994), by deletion or insertion by means of selectable markers, for instance the URA3 marker, preferably the URA3 marker from Saccharomyces cerevisiae. Alternatively, deletions, point-mutations and/or frame-shift mutations can be introduced into the functional promoters and genes required for the PDC and/or PDH activities. These techniques are disclosed, for instance, in Nature, 305, 391-397, 1983. An addition method to reduce these activities could be the introduction of STOP codons in the genes sequences or expression of antisense mRNAs to inhibit translation of PDC and PDH mRNAs.
A Kluyveromyces lactis strain wherein the PDC gene has been replaced by the URA3 gene of S. cerevisiae has already been described in Molecular Microbiology 19(1), 27-36, 1996.
The gene coding for lactate dehydrogenase may be of any species (e.g. mammalian, such as bovine, or bacterial), and it may code for the L(+)-LDH or the D(xe2x88x92)-LDH. Alternatively, both types of LDH genes may be expressed simultaneously. Further, any natural or synthetic variants of LDH DNA sequences, any DNA sequence with high identity to a wild-type LDH gene, any DNA sequence complementing the normal LDH activity may be used.
As transporter gene, for example the JEN1 gene, encoding for the lactate transporter of S. cerevisiae, can be used.
The transformation of the yeast strains can be carried out by means of either integrative or replicative vectors, linear or plasmidial.
The recombinant cells of the invention can be obtained by any method allowing a foreign DNA to be introduced into a cell (Spencer J f, et al., Journal of Basic Microbiology 28(5): 321-333, 1988), for instance transformation, electroporation, conjugation, fusion of protoplasts or any other known technique. Concerning transformation, various protocols have been described: in particular, it can be carried out by treating the whole cells in the presence of lithium acetate and of polyethylene glycol according to Ito H. et al. (J. Bacteriol., 153:163, 1983), or in the presence of ethylene glycol and dimethyl sulphoxyde according to Durrens P. et al. (Curr. Genet., 18:7, 1990). An alternative protocol has also been described in EP 361991. Electroporation can be carried out according to Becker D. M. and Guarente L. (Methods in Enzymology, 194:18, 1991).
The use of non-bacterial integrative vectors may be preferred when the yeast biomass is used, at the end of the fermentation process, as stock fodder or for other breeding, agricultural or alimentary purposes.
In a particular embodiment of the invention, the recombinant DNA is part of an expression plasmid which can be of autonomous or integrative replication.
In particular, for both S. cerevisiae and K. lactis, autonomous replication vectors can be obtained by using autonomous replication sequences in the chosen host. Especially, in yeasts, they can be replication origins derived from plasmids (2xcexc, pKD1, etc.) or even chromosomal sequence (ARS).
The integrative vectors can be obtained by using homologous DNA sequences in certain regions of the host genome, allowing, by homologous recombination, integration of the vector.
Genetic tools for gene expression are very well developed for S. cerevisiae and described in Romanos, M. A. et al. Yeast, 8:423, 1992. Genetic tools have been also developed to allow the use of the yeasts Kluyveromyces and Torulaspora species as host cells for production of recombinant proteins (Spencer J f, et al., supra; Reiser J. et al., Advances in Biochemical Engineering-Biotechnology. 43, 75-102, 1990). Some examples of vectors autonomously replicating in K. lactis are reported, either based on the linear plasmid pKG1 of K. lactis (de Lovencourt L. et al. J. Bacteriol., 154:737, 1982), or containing a chromosomal sequence of K. lactis itself (KARS), conferring to the vector the ability of self replication and correct segregation (Das S., Hollenberg C. P., Curr. Genet., 6:123, 1982). Moreover, the recognition of a 2 xcexc-like plasmid native to K. drosophilarum (plasmid pKD1-U.S. Pat. No. 5,166,070) has allowed a very efficient host/vector system for the production of recombinant proteins to be established (EP-A-361 991). Recombinant pKD1-based vectors contain the entire original sequence, fused to appropriate yeast and bacterial markers. Alternatively, it is possible to combine part of pKD1, with common S. cerevisiae expression vectors (Romanos M. A. et al. Yeast, 8:423, 1992) (Chen et al., Curr. Genet. 16: 95, 1989).
It is known that the 2xcexc plasmid from S. cerevisiae replicates and is stably maintained in Torulaspora. In this yeast the expression of heterologous protein(s) has been obtained by a co-transformation procedure, i.e. the simultaneous presence of an expression vector for S. cerevisiae and of the whole 2xcexc plasmid. (Compagno C. et al., Mol. Microb., 3:1003-1010, 1989). As a result of inter and intra molecular recombinations, it is possible to isolate a hybrid plasmid, bearing the complete 2xcexc sequence and the heterologous gene; such a plasmid is in principle able to directly transform Torulospora.
Moreover, an episomal plasmid based on S. cerevisiae AR1 sequence has also been described, but the stability of this plasmid is very low, Compagno et al. (supra).
Recently, an endogenous, 2xcexc-like plasmid named pTD1 has been isolated in Torulaspora (Blaisonneau J. et al., Plasmid, 38:202-209, 1997); the genetic tools currently available for S. cerevisiae can be transferred to the new plasmid, thus obtaining expression vectors dedicated to Torulaspora yeast species.
Genetic markers for Torulaspora yeast comprise, for instance, URA3 (Watanabe Y. et al., FEMS Microb. Letters, 145:415-420, 1996), G418 resistence (Compagno C. et al., Mol. Microb., 3:1003-1010, 1989), and cicloheximide resistance (Nakata K. et Okamura K., Biosc. Biotechnol. Biochem., 60:1686-1689, 1996).
2xcexc -like plasmids from Zycosaccharomyces species are known and have been isolated in Z. rouxii (pSR1), in Z. bisporus (pSB3), in Z. fermentati (pSM1) and in Z. bailii (pSB2) (Spencer J F. et al., supra).
Plasmid pSR1 is the best known: it is replicated in S. cerevisiae, but 2xcexc ARS are not recognized in Z. rouxii (Araki H. and Hoshima Y., J. Mol. Biol., 207:757-769, 1989).
Episomal vectors based on S. cerevisiae ARS1 are described for Z. rouxii (Araki et al., Mol Gen. Genet., 238:120-128, 1993).
A selective marker for Zygosaccharomyces is the gene APT1 allowing growth in media containing G418 (Ogawa et al., Agric. Biol. Chem., 54:2521-2529, 1990).
Any yeast promoter, either inducible or constitutive, may be used according to the invention. To date, promoters used for the expression of proteins in S. cerevisiae are well described by Romanos et al. (supra). Promoters commonly used in foreign protein expression in K. lactis are S. cerevisiae PGK and PHO5 (Romanos et al., supra), or homologous promoters, such as LAC4 (van den Berg J. A. et al., BioTechnology, 8:135, 1990) and KlPDC (U.S. Pat. No. 5,631,143). The promoter of pyruvate decarboxylase gene of K. lactis (KlPDC) is particularly preferred.
Vectors for the expression of heterologous genes which are particularly efficient for the transformation of Kluyveromyces lactis strains are disclosed in U.S. Pat. No. 5,166,070, which is herein incorporated by reference.
Pyruvate decarboxylase gene promoters, preferably from Kluyveromyces species and even more preferably from Kluyveromyces lactis, disclosed in Molecular Microbiol. 19(1), 27-36, 1996, are particularly preferred. Triose phosphate isomerase and alcohol dehydrogenase promoters, preferably from Saccharomyces species and even more preferably from Saccharomyces cerevisiae, are also preferred (Romanos et al, supra).
For the production of lactic acid, the yeast strains of the invention are cultured in a medium containing a carbon source and other essential nutrients, and the lactic acid is recovered at a pH of 7 or less, preferably at a pH of 4.5 or less, and even more preferably at a pH of 3 or less. Since the pH of the culture medium is reduced, a lower amount of neutralizing agent is necessary. The formation of lactate salt is correspondingly reduced and proportionally less regeneration of free acid is required in order to recovery lactic acid. The recovery process may employ any of the known methods (T. B. Vickroy, Volume 3, Chapter 38 of xe2x80x9cComprehensive Biotechnology,xe2x80x9d (editor: M. Moo-Young), Pergamon, Oxford, 1985.) (R. Datta et al., FEMS Microbiology Reviews 16, 221-231,1995). Typically, the microorganisms are removed by filtration or centrifugation prior to lactic acid recovery. Known methods for lactic acid recovery include, for instance, the extraction of lactic acid into an immiscible solvent phase or the distillation of lactic acid or an ester thereof. Higher yields with respect to the carbon source (g of lactic acid/g of glucose consumed) and higher productivities (g of lactic acid/l/h) are obtained by growing yeast strains, particularly Saccharomyces strains, in media lacking Mg++ and Zn++ ions or having a reduced availability of said ions. Preferably, culture media will contain less than 5 mM of Mg++, and/or less than 0.02 mM of Zn++.
The present invention offers the following advantages in the production of lactic acid:
1. When the fermentation is carried out at pH 4.5 or less, there is less danger of contamination by foreign microorganisms, as compared with the conventional process. Further, the fermentation facility can be simplified and the fermentation control can be facilitated.
2. Since less neutralizing agent is added to the culture medium for neutralization, there is correspondingly less need to use mineral acids or other regenerating agents for conversion of the lactate salt to free lactic acid. Therefore, the production cost can be reduced.
3. Since less neutralizing agent is added to the culture medium, the viscosity of the culture broth is reduced. Consequently, the broth is easier to process.
4. The cells separated in accordance with the present invention can be utilized again as seed microorganisms for a fresh lactic acid fermentation.
5. The cells can be continuously separated and recovered during the lactic acid fermentation, in accordance with the present invention, and hence, the fermentation can be carried out continuously.
6. Since the recombinant yeast strains lack ethanol production ability and pyruvate dehydrogenase activity or have both a reduced ethanol production and a reduced pyruvate dehydrogenase activity, the production of lactic acid can be carried out with higher yield in comparison to yeast strains having both a wild-type ability to produce ethanol and a wild-type ability for the utilization of pyruvate by the mitochondria.
7. The production of lactic acid by metabolically engineered non-conventional yeasts belonging to the Kluvveromyces, Torulaspora and Zygosaccharomyces species can be obtained from non conventional carbon sources (i.e., galactose-lactose-sucrose-raffinose-maltose-cellobiose-arabinose-xylose, to give some examples), growing the cells in high-sugar medium and growing the cells in presence of high concentration of lactic acid.
FIG. 1. Cloning of the lactate dehydrogenase gene shifts the glycolytic flux towards the production of lactic acid.
Key enzymatic reactions at the pyruvate branch-point are catalysed by the following enzymes: (1): pyruvate decarboxylase; (2): alcohol dehydrogenase; (3): acetaldehyde dehydrogenase;(4): acetyl-CoA synthetase; (5): acetyl-CoA shuttle from the cytosol to mitochondria; (6): acetyl-CoA shuttle from mitochondria to the cytosol; (7): heterologous lactate dehydrogenase. (8): pyruvate dehydrogenase. Enzymatic reactions involved in anaplerotic syntheses have been omitted.
FIG. 2. Diagram of the plasmid pVC1.
FIG. 3A., 3B. Diagram of the plasmid pKSMD8/7 and pKSEXH/16, respectively.
FIG. 4. Diagram of the plasmid pEPL2.
FIG. 5. Diagram of the plasmid pLC5.
FIG. 6. Diagram of the plasmid pLAT-ADH.
FIG. 7A. L(+)-Lactic acid production from the transformed Kluyveromyces lactis PM6-7a[pEPL2] during growth on Glu-YNB based media. The residual glucose concentration at T=49 was not detectable. Production of D(xe2x88x92)-lactic acid was not detectable too. The LDH specific activity was higher than 3 U/mg of total cell protein along all the experiment.
Similar results have been obtained using the bacterial L. casei LDH (data not shown). () cells/ml; (xe2x88x92) pH value; (∘) Ethanol production, g/l () L(+)-Lactic acid production, g/l
FIG. 7B. L(+)-Lactic acid production from the transformed Kluyveromyces lactis PM6-7a[pEPL2] during growth on Glu-YNB based media. Medium was buffered at time T=0 (pH=5.6) using 200 mM phosphate buffer. In this text batch, the pH value decreases much later than during the text batch shown in FIG. 7A. The residual glucose concentration at T=49 was not detectable. The LDH specific activity was higher than 3 U/mg of total cell protein along all the experiment.
Similar results have been obtained using the bacterial L. casei LDH (data not shown). () cells/ml; (xe2x88x92) pH value; (∘) Ethanol production, g/l () L(+)-Lactic acid production, g/l
FIG. 8A. L(+)-Lactic acid production from the transformed Kluyveromyces lactis PM1/C1[pEPL2] during growth on Glu-YNB based media. The residual glucose concentration at T=60 was 12,01 g/l. Longer incubation times did not yield higher productions of both biomass and L(+)-Lactic acid. The LDH specific activity was higher than 3 U/mg of total cell protein along all the experiment.
Similar results have been obtained using the bacterial L. casei LDH (data not shown). () cells/ml; (xe2x88x92) pH value; (∘) Ethanol production, g/l () L(+)-Lactic acid production, g/l
FIG. 8B. L(+)-Lactic acid production from the transformed Kluyveromyces lactis PM1/C1[pEPL2] during growth on Glu-YNB based media. Medium was buffered at time T=0 (pH=5.6) using 200 mM phosphate buffer In this text batch, the pH value decreases much later than during the text batch shown in FIG. 8A. The residual glucose concentration at T=87 was zero. The LDH specific activity was higher than 3 U/mg of total cell protein along all the experiment. () cells/ml; (xe2x88x92) pH value; (∘) Ethanol production, g/l () L(+)-Lactic acid production, g/l
Similar results have been obtained using the bacterial L. casei LDH (data not shown).
FIG. 9A. L(+)-Lactic acid production from the transformed Kluyveromyces BM3-12D[pLAZ10] cells in stirred tank bioreactor (see also text) () cells/ml; (∘) Glucose concentration, g/l () L(+)-Lactic acid production, g/l
FIG. 9B. L(+)-Lactic acid yield from the transformed Kluyveromyces BM3-12D[pLAZ10] cells in stirred tank bioreactor.
Glucose Vs lactic acid production. The yield (g/g) is 85.46%.
FIG. 10. L(+)-Lactic acid production from the transformed Torulaspora (syn. Zygosaccharomyces) delbrueckii CBS817[pLAT-ADH] during growth on Glu-YNB based media. The residual glucose concentration at T=130 was 3 g/l. Longer incubation times did not yield higher productions of both biomass and L(+)-Lactic acid. The LDH specific activity was higher than 0.5 U/mg of total cell protein along all the experiment. () cells/ml; (xe2x88x92) pH value; (∘) Ethanol production, g/l () L(+)-Lactic acid production, g/l
FIG. 11. L(+)-Lactic acid production from the transformed Zygosaccharomyces bailii ATCC60483[pLAT-ADH] during growth on Glu-YNB based media. The residual glucose concentration at T=60 was 8 g/l. Longer incubation times did not yield higher productions of both biomass and L(+)-Lactic acid. The LDH specific activity was higher than 0.5 U/mg of total cell protein along all the experiment. Similar results were obtained using a different strain (ATCC36947, data not shown) () cells/ml; (xe2x88x92) pH value; (∘) Ethanol production, g/l () L(+)-Lactic acid production, g/l